Single Nucleotide Replacement Research Articles

Альдостеронсинтаза, поліморфізм її гена CYP11B2 при артеріальній гіпертензії і асоційованих з нею кардіоваскулярних захворюваннях (огляд літератури)

В огляді надані дані зарубіжної та вітчизняної літератури щодо патогенетичної ролі альдостерону, рівнів альдостеронсинтази і поліморфізмів гена даного ферменту у хворих на різні форми артеріальної гіпертензії, при асоційованих з нею захворюваннях серця і судин, фібриляції передсердь, метаболічному синдромі, патології нирок, головного мозку. Розглядаються функції альдостерону, рівнів альдостеронсинтази і поліморфізмів гена даного ферменту в серцево-судинному ремоделюванні, можливості застосування даних маркерів для диференційно-діагностичних цілей, перспективи терапевтичного використання інгібіторів альдостеронсинтази серед різних категорій хворих з ознаками артеріальної гіпертензії.

One-step base editing in multiple genes by direct embryo injection for pig trait improvement.

The precise and simultaneous acquisition of multiple beneficial alleles in the genome is in great demand for the development of elite pig breeders. Cytidine base editors (CBEs) that convert C:G to T:A have emerged as powerful tools for single-nucleotide replacement. Whether CBEs can effectively mediate C-to-T substitution at multiple sites/loci for trait improvement by direct zygote injection has not been verified in large animals. Here, we determined the editing efficiency of four CBE variants in porcine embryonic fibroblast cells and embryos. The findings showed that hA3A-BE3-Y130F and hA3A-eBE-Y130F consistently resulted in increased base-editing efficiency and low toxic effects in embryonic development. Further, we verified that using a one-step approach, direct zygote microinjection of the CBE system can generate pigs harboring multiple point mutations. Our process resulted in a stop codon in CD163 and myostatin (MSTN) and introduced a beneficial allele in insulin-like growth factor-2 (IGF2). The pigs showed disrupted expression of CD163 and MSTN and increased expression of IGF2, which significantly improved growth performance and infectious disease resistance. Our approach allows immediate introduction of multiple mutations in transgene-free animals to comprehensively improve economic traits through direct embryo microinjection, providing a potential new route to produce elite pig breeders.

Single nucleotide replacement in the Atlantic salmon genome using CRISPR/Cas9 and asymmetrical oligonucleotide donors

BackgroundNew breeding technologies (NBT) using CRISPR/Cas9-induced homology directed repair (HDR) has the potential to expedite genetic improvement in aquaculture. The long generation time in Atlantic salmon makes breeding an unattractive solution to obtain homozygous mutants and improving the rates of perfect HDR in founder (F0) fish is thus required. Genome editing can represent small DNA changes down to single nucleotide replacements (SNR). This enables edits such as premature stop codons or single amino acid changes and may be used to obtain fish with traits favorable to aquaculture, e.g. disease resistance. A method for SNR has not yet been demonstrated in salmon.ResultsUsing CRISPR/Cas9 and asymmetrical ODNs, we were able to perform precise SNR and introduce a premature stop codon in dnd in F0 salmon. Deep sequencing demonstrated up to 59.2% efficiency in single embryos. In addition, using the same asymmetrical ODN design, we inserted a FLAG element into slc45a2 and dnd, showing high individual perfect HDR efficiencies (up to 36.7 and 32.7%, respectively).ConclusionsIn this work, we demonstrate that precise SNR and knock-in (KI) can be performed in F0 salmon embryos using asymmetrical oligonucleotide (ODN) donors. We suggest that HDR-induced SNR can be applied as a powerful NBT, allowing efficient introgression of favorable alleles and bypassing challenges associated with traditional selective breeding.

Polymorphism of CYP3A4 isoenzyme gene in patients with chronic rheumatic heart disease

AIM: This study aimed to identify the associations of CYP3A4 isoenzyme gene polymorphism with the parameters of echocardiography (EchoCG), spirometry, and endothelial function of patients with chronic rheumatic heart disease (CRHD).
 MATERIALS AND METHODS: A total of 128 patients with CRHD (15.6% men and 84.4% women) were examined. A392A, A392G, and G392G polymorphic markers were genotyped through polymerase chain reaction (PCR) with an SNP-EXPRESS electrophoretic scheme (NPF Litekh, Russia) to detect results after DNA was isolated from leukocytes in venous blood. EchoCG was implemented on an Affinity 50 apparatus (Philips, the Netherlands), endothelial function was assessed with an AngioScan01 apparatus (AngioScan-Electronics, Russia), and respiratory function was examined using a SpiroLab II spirometer (MIR Medical, Italy).
 RESULTS: The distance in a 6 min walk test did not show any significant differences among the groups: A392A 327.47 6.71 m, A392G 303.63 26.19 m, G392G 338.87 20.12 m (p=0.505). The area of the mitral opening was as follows: A392A 1.74 (1.67; 1.81) cm2, A392G 1.68 (1.45; 1.92) cm2, and G392G 1.65 (1.67; 1.81) cm2 (p = 0.214). As for the EchoCG parameters, the group of G392G homozygotes had the lowest linear dimensions of the left ventricle (the end diastolic dimension 4.83 (4.72; 4.95) cm, the end systolic dimension 2.97 (2.79; 3.14) cm, the right ventricle (2.45 [2.32; 2.58] cm), of the right atrium (4.09 [3.56; 4.62] cm), and the criteria of left ventricular hypertrophy (thickness of the interventricular septum 0.88 [0.81; 0.95] cm, and the posterior wall 0.88 (0.81; 0.95 cm). No statistically significant differences were found in the occlusion index amplitude among the groups, that is, single nucleotide replacements of CYP3A4 had no influence on the system of low-resistance arteries. Conversely, the values of the phase shift between channels (reflecting the condition of large arteries) significantly differed. The G392G polymorphism showed the worst parameters, and minimal changes were observed in the A392A group. Contour analysis demonstrated the highest augmentation index values in the G392G group, reflecting the maximal stiffness of vessels. The CYP3A4 polymorphism had no effect on the parameters of respiratory function in the studied cohort of patients. Spirometry revealed that the obstructive and restrictive parameters were not significant although homozygotes demonstrated the highest forced vital capacity of the lungs (76.5% [71.1% and 82.0%]) and forced expiratory volume for 1 s (84.6% [79.0% and 90.3%]). The maximal parameter of the vital capacity of the lungs in homozygotes for A392A (85.6% [82.3% and 88.8%]).
 CONCLUSION: Patients with CRHD homozygous for G392G had the minimum parameters of hypertrophy and dimensions of the left ventricular cavity. They also had the lowest values for the cavities of the right heart. CYP3A4 polymorphism had no effect on the parameters of respiratory function in the studied patients with CRHD.

NGS-based mtDNA Profiling Could Reveal Genetic Alterations in Schizophrenia.

Schizophrenia is a complex disease with a putative genetic background. It was hypothesized that impaired mitochondrial function due to genetic alterations in mitochondrial DNA (mtDNA) could contribute to neurological conditions, including mental disorders. The aim of the study was to find out possible pathogenic mutations and/or variants in mtDNA potentially related to schizophrenia development. The study involved 37 patients with paranoid schizophrenia, whose mtDNA profiles were compared to those of 23 healthy controls. Patients and controls were assessed using PANSS (Positive and Negative Syndrome Scale) and General Health Questionnaire (GHQ), respectively. The entire mtDNA was sequenced by the NGS platform (MiSeq®, Illumina). Bioinformatics data were processed by mtDNA Variant Processor and Analyser (Illumina), mtDNA-Server, and SPSS-17. A total of 480 mtDNA variants (single nucleotide replacements, point insertions, and deletions) were found. The polymorphic variant m.1811A>G (MT-RNR2) showed the highest frequency in schizophrenia (24.3%), as compared to the controls (4.3%) (p=0.07). Increased frequency was also found mainly in polymorphisms, belonging to complex 1 genes: MT-ND4 (11251G and 11467G), MT-ND3 (10398G), MT-ND1 (4216С), and MT-ND5 (12611G and 13708А), some of which were associated with mitochondrial dysfunction. Two individual mutations were identified in the patients: a pathogenic one - m.11778 A>G (LHON) and a newly identified, potentially pathogenic - m.4115 Т>C (NADH dehydrogenase 1). Particular mtDNA variants predominantly in complex I, probably serve as a risk genetic background in schizophrenia. The presence of pathogenic mutations in patients with psychotic manifestations expands the clinical scope of mitochondrial diseases and deserves further investigation.

ПОЛИМОРФИЗМ TOLL-ПОДОБНЫХ РЕЦЕПТОРОВ-2 У ПАЦИЕНТОВ С ХРОНИЧЕСКОЙ РЕВМАТИЧЕСКОЙ БОЛЕЗНЬЮ СЕРДЦА

Aim. To assess the contribution of TLR2 gene polymorphism to echocardiography and spirometry parameters in patients with chronic rheumatic heart disease (CRHD). Materials and Methods. A total of 128 patients with CRHD were examined – 15.6% of men and 84.4% of women. Genotyping by polymorphic markers Arg753Arg, Arg753Gln, Gln753Gln was performed by PCR with SNP-EXPRESS electrophoretic scheme (NPF Litekh, Russia) for detection of the result after extraction of DNA from leukocytes of venous blood. Echocardiography with evaluation of linear dimensions, left ventricular hypertrophy and mitral orifice area was performed on Philips Affinity 50 apparatus; respiration was evaluated using Spirolab II spirometer. Results. The distance of the 6-minute walk test in the groups did not differ significantly: (р=0.168): Arg753Arg–314.75±6.88 m, Arg753Gln–389.69±19.92 m, as well as mitral orifice area (p=0.182): Arg753Arg–1.73(1.66;1.80) cm2 and Arg753Gln–1/70(1.61;1.79) cm2. Echocardiography showed no differences in the size of the left ventricle, but in the group of Arg753Gln heterozygotes, dilatation of the left atrium was significantly higher 5.20(5.08;5.32) cm (Arg753Arg-4.98(4.83;5.13) cm) and of the right ventricle 3.10(2.90;3.30) cm (Arg753Arg-2.66 (2.59;2.72) cm) and less pronounced values of the left ventricular hypertrophy: interventricular septum thickness 1.018(0.92;1.12) cm (Arg753Arg-1.02(0.98;1.05) cm) and of the right atrium 4.40(4.10;4.70) cm (Arg753Arg-4.54(4.33;4.74) cm). In spirometry, values of both obstructive and restrictive parameters were significantly lower in the Arg753Arg homozygous group: forced lung capacity 71.04(66.15;72.94)% (Arg753Gln-84.16(79.68;88,65)%); forced expiratory volume in 1 sec 79,05(76,87;81,23)% (Arg753Gln-88,18(84,40;91.96)%); reserve inspiratory volume 84.88(81.60;88.17)% (Arg753Gln-96.45(86.73;106.18)%); reserve expiratory volume 21.29 (18.08;24.51)% (Arg753Gln-25.93(13.93;37.93)%). Conclusion. In patients with RHD, the contribution of single TLR2 nucleotide replacements to the parameters of echocardiography and spirometry is possible, which is manifested in a decrease in external respiration function in Arg753Arg homozygotes and dilatation of the left atrium and right ventricle in Arg753Gln heterozygotes.

Point mutation in the stop codon of MAV_RS14660 increases the growth rate of Mycobacterium avium subspecies hominissuis.

Mycobacterium avium subspecies hominissuis (MAH) is a pathogen that causes various non-tuberculous mycobacterial diseases in humans and animals worldwide. Among the genus, MAH is characterized by relatively slow growth. Here, we isolated a rapidly growing variant of the MAH 104 strain. The variant strain (named N104) exhibited an enhanced growth rate and higher motility compared to the parent MAH 104 strain (P104). Whole-genome sequencing analysis of N104 revealed the loss of the stop codon of MAV_RS14660 due to a single nucleotide replacement, resulting in the substitution of the codon for tryptophan. Notably, exclusion of the stop codon ligated the open reading frames and caused the fusion of two adjacent proteins. A revertant parent strain, in which a mutation was introduced to restore the stop codon, revealed that elimination of the stop codon in MAV_RS14660 was responsible for the N104 phenotype. Furthermore, we analysed the phenotypes of the parent and mutated strains by determining the functions of the MAV_RS14660 and MAV_RS14655 coding regions flanking the stop codon. The mutant strains, expected to express a fusion protein, exhibited increased resistance to antimicrobial drugs and exogenous copper toxicity compared to that of the parent strains. These findings suggest that the fusion of the MAV_RS14660- and MAV_RS14655-encoding regions in the mutant N104 strain could be related to the modified functions of these intrinsic proteins.

Molecular genetic portrait of non-small cell lung cancer in Tambov region: regional experience

8090%. The identification of gene mutation profile of non-small cell cancer allows to make a molecular portrait, which plays the crucial role in choosing the targeted therapy with tyrosine kinase inhibitors.
 The aimis to study the frequency and spectrum of activating mutations in non-small cell lung cancer in Tambov region.
 The study of the presence of mutations and the main driver gene mutation profile: EGFR, ALK, BRAF, ROS1 and PDL-1 using sections of paraffin-embedded after the surgery and biopsy in 238 patients with the help of polymerase chain reaction and immunohistochemistry. The age of patients ranged from 28 years to 82 years, with a mean age of 60.3 years.
 Results.Adenocarcinoma was detected in 80.3% of patients, and squamous cell variant in 19.7% of cases. The frequency of the driver gene activating mutations in non-small cell lung cancer was 29%, this index exceeded the frequency in the European population. Statistically significant predominance of female (63.6%) vs male (36.4%) was achieved. The mutation frequency increases with age. In this case, EGFR mutations were observed more often 22.3%. This index was slightly higher than in the European population. Statistically significant predominance of female was achieved. The highest frequency of mutations was demonstrated in patients 6570 years of age (24%). The deletion of exon 19 and single-nucleotide replacement L858R of exon 21 were observed in 54.5% and 45.5% of cases, respectively. The mutation of exon 19 was more often detected in male, at the same time the mutations of exons 19 and 21 in female were detected in almost the same frequency. Maximum frequency mutation rate (77.7%) in the EGFR gene was detected free from metastases to the regional lymph nodes. ALK mutations were found in 14.6% of patients, this index was slightly higher than the European level.
 PDL-1 expression is found in 40% male patients.
 Conclusion.The results of the study revealed some features of the profile of activating mutations in non-small cell lung cancer in the region. This data should be taken into account on planning and applying targeted therapy.

ROLE OF COL1A1 AND G2046T GENES IN UZBEKS WITH JUVENILE DYSMENORRHEA IN THE PRESENCE OF CRITERIA FOR UNDIFFERENTIATED CONNECTIVE TISSUE DYSPLASIA

Clinical and genetic examination of 118 girls was conducted, with signs of connective tissue dysplasia (СTD) - 64 and without it - 54, the control group consisted of 68 healthy girls with normal menstruation. The basis for the diagnosis of dysmenorrhea was a complaint of painful menstruation. An analysis of the distribution of allelic variants of the COL1A1 G2046T gene showed that in the group of patients with juvenile disease accompanied by СTD significantly higher compared with the control group of practically healthy individuals, only a tendency toward the reliability of alleles was observed, but they did not reach true significance. The study of the association of the COL1A1 G2046T genotype showed a significant increase in the mutant TT genotype in the group of girls with juvenile disease with СTD, compared with practically healthy individuals. Therefore, the single nucleotide variant of COL1A1 G2046T plays a role in the pathogenesis of the development of juvenile disease accompanied by СTD. This is indirectly confirmed by the indicators of oxyproline, an increase in which was significant both in comparison with practically healthy individuals and in comparison with a group of patients with juvenile disease without signs of СTD, suggests that this polymorphism is only one of the polymorphisms involved in the pathogenesis of the studied pathology. Based on the foregoing, we can conclude that the occurrence of the mutant allele of the estrogen receptor gene - alpha (single nucleotide replacement of guanine with adenine in rs2228480) is 1.9 times higher in the group of girls with juvenile dysfunction with СTD than in the group of juvenile dysfunction with СTD (χ2 = 4.515; p = 0.03). A significant difference was also found in the frequency of occurrence of type I collagen gene polymorphism (single nucleotide replacement of guanine with thymine in rs1800012) was found in girls with juvenile disease with СTD only in comparison with a group of healthy individuals (χ2 = 4.71; p = 0.03).

Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene.

The mismatch repair (MMR) system is one of the key molecular devices that prokaryotic cells have for ensuring fidelity of DNA replication. While the canonical MMR of E. coli involves 3 proteins (encoded by mutS, mutL and mutH), the soil bacterium Pseudomonads putida has only 2 bona fide homologues (mutS and mutL) and the sensitivity of this abridged system to different types of mismatches is unknown. In this background, sensitivity to MMR of this bacterium was inspected through single stranded (ss) DNA recombineering of the pyrF gene (the prokaryotic equivalent to yeast's URA3) with mutagenic oligos representative of every possible mispairing under either wild-type conditions, permanent deletion of mutS or transient loss of mutL activity (brought about by the thermoinducible dominant negative allele mutLE36K ). Analysis of single nucleotide mutations borne by clones resistant to fluoroorotic acid (5FOA, the target of wild type PyrF) pinpointed prohibited and tolerated single-nucleotide replacements and exposed a clear grading of mismatch recognition. The resulting data unequivocally established the hierarchy A:G < C:C < G:A < C:A, A:A, G:G, T:T, T:G, A:C, C:T < G:T, T:C as the one prevalent in Pseudomonas putida. This information is vital for enabling recombineering strategies aimed at single-nucleotide changes in this biotechnologically important species.

Creation of Cybrid Cultures Containing mtDNA Mutations m.12315G>A and m.1555G>A, Associated with Atherosclerosis.

In the present work, a pilot creation of four cybrid cultures with high heteroplasmy level was performed using mitochondrial genome mutations m.12315G>A and m.1555G>A. According to data of our preliminary studies, the threshold heteroplasmy level of mutation m.12315G>A is associated with atherosclerosis. At the same time, for a mutation m.1555G>A, such a heteroplasmy level is associated with the absence of atherosclerosis. Cybrid cultures were created by fusion of rho0-cells and mitochondria from platelets with a high heteroplasmy level of the investigated mutations. To create rho0-cells, THP-1 culture of monocytic origin was taken. According to the results of the study, two cybrid cell lines containing mutation m.12315G>A with the heteroplasmy level above the threshold value (25% and 44%, respectively) were obtained. In addition, two cybrid cell lines containing mutation m.1555G>A with a high heteroplasmy level (24%) were obtained. Cybrid cultures with mtDNA mutation m.12315G>A can be used to model both the occurrence and development of atherosclerosis in cells and the titration of drug therapy for patients with atherosclerosis. With the help of cybrid cultures containing single nucleotide replacement of mitochondrial genome m.1555G>A, it is possible to develop approaches to the gene therapy of atherosclerosis.

PB2066 DAMAGE OF THE TP53 GENE POLYADENYLATION SIGNAL IN DLBCL

Background:Single nucleotide replacement rs78378222 lead to change of the normal TP53 gene polyadenylation signal and cause disorders of processing of 3′‐end of mRNA. The experimental data have suggested that rare allele C of the rs78378222 leads to much lower level of TP53 expression and decrease the apoptosis induction level in cells under the influence of genotoxic agents in comparison with allele A. Thus, rs78378222 is thought to be the unique allele variant of TP53 gene with the reduced p53 function.Aims:The purpose of the present study was to reveal the frequency of the rs78378222 of TP53 in tumor tissue of the patients with diffuse large B‐cell lymphoma (DLBCL).MethodsGenomic DNA was isolated from formalin‐embedded paraffin blocks of lymph nodes and extranodal tumor lesions biopsies of 92 patients with DLBCL by phenol‐chloroform extraction method using guanidine. The tissue sections containing at least 70‐80% of the tumor cells were taken. Genotyping of the rs78378222 was performed by the PCR‐RFLP method. To confirm the presence of rare allele C of the rs78378222 Sanger's direct sequencing was performed.Results:The frequency of the rs78378222 in DLBCL tumor tissue was 9/92 (9.8%). It was noted that 5 of 9 patients had minor allele C in the homozygous state. Since the detection of cases of the rare homozygous genotype C/C of the rs78378222 in normal tissue has not described previously, the received results indicated a loss of heterozygosity in the TP53 gene in DLBCL tumor tissue of rs78378222 carriers. Only two of these cases were combined with mutations in the gene. In three cases ‐ loss of heterozygosity was the only aberration in TP53.Summary/Conclusion:Aberrations in 3′‐UTR of TP53 gene may be a universal carcinogenesis mechanism, playing role in pathogenesis of DLBCL as well. The loss of normal allele A rs78378222 stimulates the significant growth of cells malignant potential.

Engineering of high-precision base editors for site-specific single nucleotide replacement

Engineering of high-precision base editors for site-specific single nucleotide replacement

Engineering of high-precision base editors for site-specific single nucleotide replacement

RNA-guided nucleases of the CRISPR/Cas type can be repurposed as programmable nucleotide deaminases to mediate targeted nucleotide substitutions. Such base editors have enormous potential in genome editing, gene therapy and precision breeding. However, current editors suffer from limited specificity in that they edit different and/or multiple bases within a larger sequence window. Using cytidine deaminase base editors that elicit C-to-T mutations, we show here that high editing precision can be achieved by engineering the connection between the deaminase domain and the Cas domain of the editor. By systematically testing different linker sequences and removing non-essential sequences from the deaminase, we obtain high-precision base editors with narrow activity windows that can selectively edit a single cytidine at a specific position with high accuracy and efficiency. These base editors will enable the use of genome editing in applications where single-nucleotide changes are required and off-target editing of adjacent nucleotides is not tolerable.

Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system

BackgroundThe soil bacterium Pseudomonas putida KT2440 is a “generally recognized as safe”-certified strain with robust property and versatile metabolism. Thus, it is an ideal candidate for synthetic biology, biodegradation, and other biotechnology applications. The known genome editing approaches of Pseudomonas are suboptimal; thus, it is necessary to develop a high efficiency genome editing tool.ResultsIn this study, we established a fast and convenient CRISPR–Cas9 method in P. putida KT2440. Gene deletion, gene insertion and gene replacement could be achieved within 5 days, and the mutation efficiency reached > 70%. Single nucleotide replacement could be realized, overcoming the limitations of protospacer adjacent motif sequences. We also applied nuclease-deficient Cas9 binding at three locations upstream of enhanced green fluorescent protein (eGFP) for transcriptional inhibition, and the expression intensity of eGFP reduced to 28.5, 29.4, and 72.1% of the control level, respectively. Furthermore, based on this CRISPR–Cas9 system, we also constructed a CRISPR–Cpf1 system, which we validated for genome editing in P. putida KT2440.ConclusionsIn this research, we established CRISPR based genome editing and regulation control systems in P. putida KT2440. These fast and efficient approaches will greatly facilitate the application of P. putida KT2440.

The reversion of A23525T of polymorphism of FTO gene in persons with overweight in conditions of hypocaloric diet

ObjectiveTo study the association of A23525T polymorphism of the FTO gene with overweight/obesity and the possibility of reversion of single-nucleotide replacements rs9939609 in a hypocaloric diet. Materials and methodsPolymorphisms of the FTO gene (A23525T, rs9939609) are typified by the SNP method (single nucleotide polymorphism) with allele-specific primers on the test systems of the NPF "Liteh".The frequencies rs9939609 were studied in persons with overweight, I–III degree of obesity (n = 84) compared with the control group (n = 32). The reversion of polymorphic variants of genes under the influence of a hypocaloric diet has been analyzed. The experimental data are processed in SPSS Statistics 22.0. ResultsIn the pilot study, unlike previously published studies, a reliable association of the genotype of the FTO gene with a violation of lipid metabolism (OR = 2.33, CI 95%) was shown not by A23525A, but by T23525T. A positive correlation was established between the mean force between the TT genotype and the weight of the participants in the experiment (r = + 0.625, p = 0.022), fat mass (r = + 0.610; p = 0.046), body mass index (BMI) (r = + 0.577; p = 0.039) and the circumference of the hips (r = + 0.636; p = 0.039). In 25 out of 84 participants (29.7%) after RTD, a partial or complete reversal (reversal) of nucleotides occurred in rs9939609: 40% of them were mutations transversion caused by the replacement of T→A nucleotides and the A23525T, T23525T transition of genotypes into the homozygous A23525A variant. ConclusionThe TT genotype of the FTO gene is associated with obesity. The reversal of polymorphisms in the FTO gene under the influence of RDT is mainly associated with transversion of nucleotides (А→Т) at the corresponding site (23525) of the intron mutation (rs9939609).

Refactoring the Genetic Code for Increased Evolvability.

ABSTRACTThe standard genetic code is robust to mutations during transcription and translation. Point mutations are likely to be synonymous or to preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best-performing mutant requires replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter a single nucleotide per mutation event, due to the extreme rarity of adjacent mutations. In this theoretical study, we suggest a radical reordering of the genetic code that maximizes the mutagenic potential of single nucleotide replacements. We explore several possible genetic codes that allow a greater degree of accessibility to the mutational landscape and may result in a hyperevolvable organism that could serve as an ideal platform for directed evolution experiments. We then conclude by evaluating the challenges of constructing such recoded organisms and their potential applications within the field of synthetic biology.

GENEBANK ANALYSIS: SINGLE NUCLEOTIDE POLYMORPHISMS OF ANIMALS MITOCHONDRIAL GENOME UKRAINIAN GRAY AND UKRAINIAN WHITEHEAD CATTLE BREEDS

Examination of variation in mitochondrial DNA (mtDNA) control region sequences has been pivotal in the elucidation of bovine phylogeography. Initial studies have demonstrated a deep bifurcation in bovine mtDNA phylogeny, which indicates a predomestic divergence between the two major taxa of cattle, humped zebu (Bos indicus) and humpless taurine (Bos taurus). Subsequent genetic investigations have yielded further inference regarding origins within the B. taurus lineage. B. taurus mtDNA sequences fall into one of five ancestral star-like haplotypic clusters, which are geographically distributed. Just one of these clusters, T3, predominates in Western Europe. Symmetrically, diversity within Africa is composed almost exclusively of members of a separate haplotypic cluster, T1, which is rarely detected elsewhere. The almost mutually exclusive geographic distribution of these two haplotypic clusters allows geographical exceptions to be securely identified as secondary introductions. We investigated a comparative analysis of mitochondrial genome sequences for different breeds of cattle (Bos taurus, Bos indicus) with global genetic bank. Mitochondrial DNA sequences from bovine animals (Bos taurus) breeds Ukrainian Whitehead and Ukrainian Gray freely available on the global genetic bank (http://www.ncbi.nlm.nih.gov/Genebank/). Local alignment of sequences for mitochondrial genome of different cattle breeds was performed using the program MEGA 4.0. For the detection of nucleotide replacements used mitochondrial DNA sequence of Bos taurus Hereford breed (Anderson S. at al., 1982) as a reference (accession number V00645). Here we report the analysis results of testing for 9 genotypes Ukrainian Gray mitochondrial DNA sequences showed that one animal (GQ129208) has haplotype Bos indicus, other belongs to haplogroup T1 with European origin mtDNA. Analysis of single nucleotide replacement in one of the hypervariable regions mtDNA (position number 16019-16339) shows, that among 10 submitted genotypes of Ukrainian Whitehead the 3 of them (FJ014303, FJ014298, FJ014294) relating to T1a mtDNA haplogroup of African origin, which characterized by replacement of T to C at position 16255. Also have been two animals (FJ014301, FJ014295) with single nucleotide replacements with relatives to Bos indicus mtDNA haplogroup. We performed alignment with reference sequences (Bos_taurus_v00654.1) and comparative nucleotide sequences analysis of another hypervariable D-loop (position number 1-240) mtDNA with 5 Ukrainian Whitehead genotypes and 5 Ukrainian Gray genotypes represented in genetics bank. Among the Ukrainian Whitehead genotypes (FJ014298, FJ014297, FJ014296, FJ014295, FJ014294) all were polymorphic that characterizes large differentiation these animals for maternal and describe deep heterogeneous parent population of studied group. We determined one animal with genotype FJ014295 was significantly different by the number of segregation sites. The analyzed sequences (FJ014290, FJ014289, FJ014288, FJ014287, FJ014286) of 5 Ukrainian Gray genotypes showed no polymorphism in hypervariable D-loop (position number 1-240) mtDNA. The mtDNA analysis of different species of animals allowed to distribute their mtDNA belonging to European, African and Asian haplogroups. The technique, which allows to differentiate the animals represented by their belonging to the respective haplogroups. The process that gave rise to different genotypes in one lineage is clearly of fundamental importance in understanding intraspecific mitochondrial polymorphism and evolution in mammals. Сomprehensive study genetic material provide more opportunities to optimize costs in-situ conservation of different cattle breeds, to optimize methods and techniques which used in ex-situ conservation programmes of National gene bank of animal genetic resources.

HLA-B*13:95, a novel variant of HLA-B*13, discovered in a Taiwanese blood donor.

A single nucleotide replacement at residue 430 of HLA-B*13:01:01 results in a new allele, HLA-B*13:95.

The interpopulation differences between nucleotide sequence polymorphisms in alleles of the STR-loci of human chromosomal DNA

The objective of the present work was to study the phenomenon of nucleotide sequence polymorphism in alleles of the STR-loci of human chromosomal DNA and to estimate its interpopulation differences with a view to the search for the molecular-genetic markers to be used as an efficient tool for the determination of belonging of the subjects of interest to a given population. We undertook the comprehensive analysis of amplified DNA fragment sequence polymorphism (AFSP) and amplified DNA fragment length polymorphism (AFLP) with the use of the PLEX-ID-TM analytical mass-spectrometry platform (Abbott Molecular, USA). The interpopulation differences were estimated in terms of the presence or the absence of single nucleotide replacements (SNP) in the STR markers based on a few population samples. Some of the loci of interest were found to have allelic variants the occurrence of which was significantly different in individual samples. Such alleles are of importance for the further investigation since they can be regarded as potential ethno-geographical markers. Their application opens up the new promising prospects for the expert detection of the ethnic affiliation of individual subjects.